FIELD OF INVENTION
The present invention relates to a method of and apparatus for detecting microorganisms. More particularly, it relates to a microorganism detecting method and apparatus for identifying microorganisms contained on a microorganism sample slide, such as a sample in which the microorganisms are contained in a smear of a body fluid on a microscope slide subjected to fluorescent staining.
Heretofore, microbial tests in utilized in quality control in the manufacturing process of food, beverages, pharmaceuticals and other consumables have relied upon the results of cultures requiring various media, ambient conditions, and other assorted growth requirements. Typically the time required for adequate growth of these cultures is from 48-72 hours. The reliance upon the measurable growth in these cultures by definition delays the process of evaluating the safety of the goods in question, which in turn delays getting products to the consumer, and also increases the cost to the manufacturers as a result of having to hold large inventories for extended periods before being able to release the goods to market.
In view of such circumstances, a large number of rapid measurement methods have heretofore been devised (Misao HARUTA, et el. Simplification. Automation and Speed-up of Food-microbiological Tests. p. 11. Science Forum (1985), and Toshiki MORICHI. New Food Industry. 49 (1989)), but satisfactory methods or apparatuses have not been developed yet. There have been developed. for example an ATP measurement method (Molin, O}. Milsson, L. and Ansehn. S. J. Clin. Microbiol., 18.521 (1983)). an impedance method {Brown. D., Wamer. M. Taylor. C. and Warren, R., J. Clin. Pathol. 37. 65-69 (1984)). an enzyme/fluorescence detecting method (Japanese Patent Laid-open Pub. No. 116700/1983), and a DEFT method in which a membrane filter method and a fluorescence microscope method are combined (G. L. PETTIPHER, UBALDINAM and RODRIGUES, J. Appl. Bacterol., 53. 323 (1982)). and apparatuses to which these principles are applied are commercially available. However, problems are still left in points of accuracy and rapidity. More specifically, the rapid measurement methods presently in practical use exhibit, at the utmost, accuracies of 102-104microorganisms/mi. One day is usually required for one microorganism to reach this germ density. Another difficulty is that the running costs of culture grounds, reagents etc. are high.
Recently, an apparatus wherein microorganisms are automatically detected rapidly by a fluorescence detecting method subjecting the microorganisms to fluorescent staining has been proposed (Japanese Patent Laid-open Pub. No. 53447/1988). Studies have been done to put the apparatus into practical use as ones of the more rapid instruments to detect microbes. Microscopic foreign matter, has auto-fluorescence and exist in significant concentration in the natural world. The above mentioned apparatus simultaneously detects both foreign matters and microorganisms. Consequently, in the test of microorganisms contained in a sample to be inspected, such as meat, foul or beverage the problem of accurate assessment of true concentration of microbial contamination cannot be resolved by visual inspection under a fluorescence microscope. Accordingly, the most important issue to resolve in providing an automated method and apparatus for detecting fluorescing microorganisms is the accurate discrimination between microbes and artifacts.
The prior art offered by Niwa et al., in U.S. Pat. No. 5,480,804 addresses a method of and apparatus for detecting microorganisms. Like the current invention, it too suggest the value of fluorescent staining the microbes, but offers a means of detection that depends on the photoelectric conversion fluorescence of a specified wavelength while filtering out noise ascribable to the autofluorescence of artifacts, and basically automatically maps the signal outputs representing the microbes on the sample, without any reliance on imaging microorganisms.
The closest known prior art is called the Dynamic Microscope Image Processing Scanner (DMIPS), U.S. Pat. No. 4,700,298. This device is classified as an image scanner which relies upon a microscope. Unlike its predecessor, the present invention uses a proprietary integrated optical/electronic system for image acquisition rather than a standard microscope. Unlike it predecessor, the invention uses a computer driven autofocus mechanism using a high precision stepping motor to drive the optical/electronic image sensor in the z axis that is integrated with the x,y plane displacement for scanning provided by the precision motorized stage.
(DMIPS) utilizes an analog-digital converter in its image sensor, rather than a simple digital system with no analog components. Image processing in the prior art (DMIPS) relies upon edge detecting algorithms, whereas the current invention uses filters to eliminate all signals but those produced by the desired objects for analysis. In addition, unlike the prior art, the current invention utilizes a trained knowledge based software engine supplied with a complete image data base of microorganisms as well as artifacts, which permits the current invention to actually quantitate and confirm the numbers and positions of microbes detected within a sample utilizing actual images of the microbes. The prior art does not include a robotic specimen handling mechanism, automated changeable light sources, automated lens changing capability, automated condenser operation or automated excitation and emission filter assemblies, nor automated archiving of results.